The primary objective of the proposed studies is the ongoing elucidation of the diverse molecular neuro-immune mechanisms by which the neuropeptide substance P (SP) modulates the responses of lymphocyte cell lines transfected with the SP receptor (SPR) cDNA and of lymphocytes that constitutively express the SPR that have been isolated from embryonic and adult rat thymus, spleen and intestinal Peyer's Patches (PP). SP is present at elevated concentrations in tissues at sites of inflammation and has been implicated as a significant mediator contributing to both acute and chronic inflammatory processes. The specific aims of this competitive renewal is to continue to test the hypothesis that SP is an important immunological mediator with an integrated and comprehensive assessment of the mechanisms by which SP regulates lymphocyte function. this will be accomplished with three specific aims: 1) the PI will examine the localization and anatomic relationship of SPR(+) cells and SP containing neurons in defined immune tissues (spleen, thymus, PP) in embryonic and adult rats. Immunocytochemistry with an immunoaffinity purified Fab antibody raised against a full length SPR fusion protein generated in bacteria will be used, as will in-situ hybridization and RNase protection assays; 2) the immunologic phenotype and functional responses to well characterized stimuli of SPR(+) lymphocytes from the aforementioned tissues at various stages of development will be studied in detail. These cells will be isolated using the SPR Fab antibody and cell sorting techniques. Proliferative and synthetic (cytokine production) responses will be examined. The phenotype of SPR(+) cells will be defined with well characterized antibodies to rat surface antigens; 3) using lynphocyte cell lines transfected with the rat SPR epsilonDNA (Jurkat and Reh) and SPR(+) lymphocytes isolated from the above tissues, we will examine the effects of SPR stimulation on the expression and function of cell surface molecules that play an important role in neurogenic inflammation such as cellular activation, adhesion and neuropeptide degradation. The PI has developed assays to answer the above questions, and the expected results and anticipated problems have been carefully analyzed. These three specific aims form the basis for a focused yet integrated approach to our ongoing examination of the hypothesis that SP is an important immunological mediator.